Deciphering the mechanism of MYcolic acids TRAnsport by MmpL3, a highly valuable drug target for the treatment of tuberculosis – MyTraM

To achieve its objectives, the project relied on an integrated set of modern biochemical and structural biology techniques:

Recombinant protein expression in E. coli to produce large quantities of purified MmpL3 from various mycobacterial species.

Protein purification using affinity, ion-exchange, and gel-filtration chromatography to obtain high-quality samples.

Structural determination through X-ray crystallography, including testing crystals at synchrotron facilities.

Implementation of peptidiscs, an innovative method that stabilizes membrane proteins in detergent-free “nanodiscs,” greatly improving imaging quality.

Cryo-electron microscopy (cryo-EM) in collaboration with specialized facilities to visualize MmpL3 and related proteins at near-atomic resolution.

Computational modeling and docking to explore how inhibitors interact with MmpL3.

Collaborative biochemical assays (including in Mycobacterium smegmatis cells) to confirm MmpL3 as the direct target of newly discovered antitubercular compounds.

These complementary methods built a robust experimental framework for studying MmpL3 and its partners with unprecedented precision

Over the course of 43 months, the project achieved several key milestones:

Successful purification and crystallization of MmpL3 from various mycobacterial species, including M. smegmatis and M. abscessus.

First three-dimensional structures of MmpL3 obtained by X-ray crystallography, and the high-resolution structure of its partner protein TtfA, essential for transport.

Development of new purification techniques, including the innovative use of “peptidiscs”, which stabilize membrane proteins without detergents — a breakthrough for future structural studies.

Validation of MmpL3 as a direct drug target for promising antitubercular compounds (such as PIPD1), through biochemical, microbiological, and computational approaches.

Establishment of a robust experimental framework for studying the broader MmpL protein family, laying the groundwork for future drug discovery efforts.

The project resulted in 9 scientific publications with 4 directly related to the projet and 5 other through collaboration involving people involved in the Mytram projet. An international collaborations with teams in Denmark was estsblihed. Further a doctoral thesis was achieved through this research program.

A distinctive feature of the MyTram project is its technical innovation in studying difficult membrane proteins. The use of peptidiscs, advanced crystallography, and cryo-electron microscopy approaches has overcome several long-standing barriers in the field.

These advances not only benefit tuberculosis research but also open new perspectives for studying similar transport systems in other bacteria, including those relevant to antimicrobial resistance.

Aftr the Mytram project finished the team succesfully unlock several of the objectives notably :

Refine the cryo-EM structure of MmpL3 at higher resolution through the peptidisc technology (Couston et al., 2023)

Investigate the functional interactions between MmpL3 and its partners (such as TtfA) (PhD Thesis manuscript Julie Couston 2025)

The MyTram project thus marks a significant step forward in understanding how tuberculosis bacteria build their outer membrane, providing a strong foundation for future therapeutic breakthroughs.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), is one of the deadliest human pathogen. Despite existing chemotherapy, Mtb has been responsible for the death of 1.4 million people and about 10 million new infections in 2015 (WHO report on TB, 2016). This concerns not only developing countries since 5000 new cases are reported yearly in France. The current problems in TB eradication are: lengthy treatments, co-infection with HIV and emergence of multidrug-resistant strains of Mtb. During the last 50 years, very few antitubercular drugs have been discovered and put on the market. Therefore, identifying new molecules targeting mycobacteria is urgent although highly challenging. Most mycobacteria are naturally resistant to antibiotics for which the highly hydrophobic cell wall represents an impermeable barrier. Mycolic acids (MA) are very long lipids made of 90 carbon atoms that are essential components of the mycomembrane and contribute to the high hydrophobicity of the cell wall. MA are synthesized in the cytoplasm and then transported to the periplasm by a specific transporter, MmpL3, which belongs to the superfamily of Resistance-Nodulation-Division permeases. To date, our knowledge about the mechanism by which MA are transported by MmpL3 remains very limited, due to the lack of both in vitro characterization and structural information. The fact that MmpL3 is essential for mycobacterial growth makes it an extremely attractive drug target for future translational applications. Recent whole-cell-based screening conducted by several independent teams, including ours, led to the identification of various chemical entities exhibiting potent antitubercular activity. The mode of action of all these chemotypes involves the inhibition of MA transport to the bacterial surface. In most studies, MmpL3 was designated as the primary target based on the presence of mutations occurring in mmpL3 in spontaneous resistant strains. Among them, some have already reached phases II or III of clinical trials and/or have shown to exhibit synergetic effects with existing antitubercular drugs. Despite these exciting promises, concerns have recently been raised regarding the real implication of MmpL3 as the target of many of these compounds as well as their mechanism of action. Therefore, describing, at a molecular and structural level, the MmpL3-mediated transport mechanism might help to understand how MA are translocated to the cell surface and to validate the importance of MmpL3 in cell wall assembly. This may also greatly help to describe the mode of action of some of the recently identified MmpL3 inhibitors.
The objectives of MyTraM consists of the 1) expression and purification of large amounts of recombinant MmpL3; 2) implementation of hybrid structural biology approaches including X-ray crystallography and cryo-electron microscopy to determine the three-dimensional structure(s) of MmpL3; 3) development of an innovative biochemical in vitro assay to assess MA transport and to investigate the mode of action of several MmpL3 inhibitors; and 4) determination of the binding constants of MmpL3 substrates and inhibitors in solution using microscale thermophoresis.
We anticipate that this 3-year project should add important breakthroughs in our understanding of MmpL structure-function relationships and lead to a more precise description of the mode of inhibition of a family of promising anti-TB compounds. On a longer term, these studies should also aid in the future improvement of already existing MmpL3 inhibitors and in the conception of new generations of MmpL3-based drugs for the treatment of TB and other mycobacterial infections.